microRNA-338-3p suppresses lipopolysaccharide-induced inflammatory response in HK-2 cells

Background Inflammation is the most common cause of kidney damage, and inflammatory responses in a number of diseases are mediated by microRNA-338-3p (miR-338-3p). However, there are only a few reports which described the regulation of miR-338-3p in human proximal tubular cells. The goal of this study was to see how miR-338-3p affected lipopolysaccharide (LPS)-caused inflammatory response in HK-2 cells. Methods LPS was used to construct an inflammatory model in HK-2 cells. miR-338-3p mimic was used to increase the levels of miR-338-3p in HK-2 cells. MTT, JC-1 staining, and apoptosis assays were used to detect cell viability, mitochondrial membrane potential (MMP), and apoptosis, respectively. The production of inflammatory factors and the levels of p38, p65, phospho-p65, phospho-p38, Bax, Bcl-2, cleaved caspase-9, and cleaved caspase-3 were investigated using real-time polymerase chain reaction, western blotting, or enzyme-linked immunosorbent assay. Results The levels of miR-338-3p were significantly lower in serum from patients with sepsis-induced kidney injury compared to the serum from healthy volunteers (P < 0.05). LPS reduced the level of miR-338-3p in HK-2 cells (P < 0.05). HK-2 cell viability, mitochondrial membrane potential, and Bcl-2 mRNA and protein levels were decreased by LPS (all P < 0.05). Apoptosis, the mRNA and protein levels of inflammatory cytokines (IL-1β, IL-6, IL-8, and TNF-α) and Bax, and the levels of cleaved caspase-9 and caspase-3 were increased by LPS (all P < 0.05). Raising the level of miR-338-3p mitigated these effects of LPS (all P < 0.05). Conclusion LPS-induced inflammation in HK-2 cells is reduced by miR-338-3p. Supplementary Information The online version contains supplementary material available at 10.1186/s12860-022-00455-0.


Introduction
Sepsis is caused by the dysfunctional response of the host to infection, which produces a large number of inflammatory factors. Acute kidney injury (AKI) is a common complication among hospitalized patients with sepsis, is associated with increased mortality [1,2], and is characterized by endothelial injury with hemodynamic dysfunction [3]. Inflammatory cytokines (IL-1β, IL-6, IL-8, TNF-α) produced by the proximal tubular epithelial cells of the damaged kidney, penetrate into the renal interstitium and further damage renal function; the resultant damage indicates that the pathogenesis of AKI is complicated by inflammation [4][5][6]. Oxidative stress, together with inflammation, can accelerate the decline of renal function [7,8]; therefore, the exploration of inflammatory responses in renal injury will contribute to the treatment and improve the survival rate of patients with this condition. The NF-κB (p65) and MAPKs (p38) *Correspondence: 13599040300@139.com signaling pathways are the main pathways that regulate the production of inflammatory cytokines [9], so the activation of these pathways will improve the production of inflammatory cytokines. Furthermore, sepsis-induced AKI is considered to be a comprehensive response, including transcriptional events, mitochondrial activity, and apoptosis [3].
MiRNA-338-3p (miR-338-3p) is located in the seventh intron of the apoptosis-associated tyrosine kinase gene [17]. Apoptosis is the main mode of cell death, the decrease of mitochondrial membrane potential is the early activity of apoptosis, and the dysregulation of apoptosis-related proteins (Bcl-2, Bax, cleaved caspase-9, and cleaved caspase-3) plays an important role in the occurrence and development of apoptosis [18]. Therefore, the investigation of the relationship between miR-338-3p and apoptosis-related proteins (Bcl-2, Bax, cleaved caspase-9, and cleaved caspase-3) is helpful to reveal the molecular mechanism of miR-338-3p's regulation of inflammatory damage in renal cells. The level of miR-338-3p is decreased in virusinduced neurodegenerative diseases [19]. miR-338-3p can alleviate inflammatory damage. For example, miR-338-3p inhibits inflammation in acute liver injury caused by N-acetyl-p-aminophenol, and relieves LPSinduced inflammatory damage in 16HBE cells (human bronchial epithelioid cells) [20,21]. However, it is unknown what role miR-338-3p plays in renal injury.
In this study, HK-2 cells were stimulated by LPS to establish an in vitro model of inflammatory damage. HK-2 cells overexpressing miR-338-3p were treated with LPS, and then cell viability, the secretion of inflammatory cytokine, mitochondrial membrane potential (MMP) changes, and apoptosis were detected to study the role of miR-338-3p in LPS-induced inflammation.

Cell culture
The HK-2 cell line, which is an epithelial cell line of the proximal convoluted tubule of the human renal cortex, was purchased from Xiamen Immocell Biotechnology Co., Ltd. (Catalog number: IM-H060). DMEM with 10% fetal bovine serum and 5ng/ml epidermal growth factor was used to culture the cells. The cells were incubated at 37 °C, with 5% carbon dioxide, and 70-80% humidity.

Grouping of cells
Dimethyl sulfoxide (DMSO), LPS, LPS + mimic negative control (NC), and LPS + miR-338-3p mimic groups designed with HK-2 cells in 6-well plates. In the DMSO group, HK-2 cells were treated with complete medium supplemented with 0.1% DMSO (as a negative control) for 24 h. In the LPS group, HK-2 cells were treated with 5 µg/mL LPS for 24 h. LPS + mimic NC group and LPS + miR-338-3p mimic group were transfected with 200 pmol negative control of miR-338-3p mimic (mimic NC) and miR-338-3p mimic for 24 h, respectively, and then the cells were treated 5 µg/mL LPS. After 24 h, cells or cell supernatants were collected for subsequent experiments.

Blood sample collection
The Fujian Maternity and Child Health Hospital Ethics Committee approved this study (approval number: 2022KD0133), which was carried out in compliance with the Helsinki Declaration. Informed consent papers were signed by all volunteers. Vacuum vascular collection was used to collect peripheral venous blood (5 mL) from all volunteers in the morning while they were fasting. The blood was centrifuged to isolate the serum which was stored at -80 °C for further analysis.

Real-time polymerase chain reaction (RT-PCR)
To extract RNA, cells were treated with TRI reagent ® (Sigma-Aldrich, Catalog number: T9424) and serum was treated with serum miRNA isolation kit (TIAN-GEN, Catalog number: DP503) according to the manufacturer's instructions. The obtained RNA was reverse transcribed with the PrimeScript RT Reagent Kit (Takara, catalog number: RR047A). Reverse transcription of the miRNAs was completed using specific primers (Table 1). RT-PCR was performed using the reverse transcriptional RNA, Agilent-Strata gene MxReal-Time qPCR system, and SYBR Green Master Mix (VAZYME, catalog number: Q111-02). The thermocycling conditions of qPCR were 95 °C for 5 min, followed by 40 cycles at 95 °C for 10 s and 60 °C for 30 s. The relative  Table 1.

MTT assay
To explore the effects of LPS and miR-338-3p on cell viability, we used MTT assay to detect cell viability. The treated HK-2 cell was seeded in 96-well plate at 1 × 10 4 per well. After 12 h, 5 mg/mL MTT (10 µL per well) was added and incubated at 37 °C for 4 h. Then, we discarded the culture medium and added 150 µL DMSO to each well. The SpectraMax Absorbance Reader (Molecular Devices, San Francisco, CA, USA) was used to measure the absorbance at 490 nm.

Detection of mitochondrial membrane potential (MMP)
After the treated cells were obtained, MMP was detected using JC-1 staining assay kit (Beyotime, catalog number: C2006) as directed by the manufacturer. The cells were then examined by flow cytometry (ACEA Bioscience Inc.) at Ex/Em = 549/575 nm.

Apoptosis assay
Subsequent to treatment with the indicated compounds, HK-2 cells were collected to analyze apoptosis using an apoptosis detection kit (Vazyme, catalog number: A211-01) as directed by the manufacturer. Flow cytometry (ACEA Bioscience Inc.) was used to detect and analyze cell apoptosis.

Statistical analysis
The difference between two groups or among multiple groups was assessed using Student's t-test (unpaired) or analysis of variance (ANOVA) in SPSS software (version 22.0), respectively. A difference of P < 0.05 was considered significant. GraphPad Prism 8.2.1 was used to obtain the graphs.

LPS negatively regulates miR-338-3p
To investigate the effect of LPS on miRNA, we used 5 µg/ mL LPS to stimulate HK-2 cells for 24 h, and RT-PCR was used to detect the levels of miRNAs in HK-2 cells. The results showed that in LPS-stimulated cells, the levels of miR-326, miR-16-5p, miR-30c-5p, miR-338-3p, miR-548, and miR-599 were significantly decreased (Fig. 1A). Subsequently, we discovered that the decrease of miR-338-3p in HK-2 cells occurred with the increase of LPS dose or the prolongation of stimulation time (Fig. 1B, C). Moreover, patients with sepsis-induced kidney injury had significantly higher levels of miR-338-3p in their blood than healthy volunteers (Fig. 1D). LPS inhibited miR-338-3p expression in HK-2 cells, according to these findings.

LPS suppresses cell survival by targeting miR-338-3p
LPS affects the survival of cells by activating inflammatory response in the cells [6,12]. The viability of HK-2 cells was determined using the MTT assay after they were stimulated with various concentrations of LPS or 5 g/mL LPS for various time periods. The results verified that the survival of cells was impaired by LPS, and the higher the amount of LPS, or the longer the LPS action time, the lower the survival rate of cells ( Figs. 2A,   B). The HK-2 cells were simultaneously treated with LPS and supplemented with miR-338-3p, and the levels of miR-338-3p and cell survival rate were detected. The results suggested that LPS reduced miR-338-3p level, but miR-338-3p level was increased when the HK-2 cells were treated with LPS and transfected with the miR-338-3p mimic (Fig. 2C). In addition, miR-338-3p diminished the LPS-induced decrease in cell survival (Fig. 2D). These data indicated that LPS impaired cell survival by inhibiting miR-338-3p expression.

Overexpression of mir-338-3p relieves LPS-induced apoptosis
According to previous report [22], LPS induces apoptosis, and the aforementioned results show that LPS targets miR-338-3p; therefore, it is possible that LPS promotes apoptosis by targeting miR-338-3p. We performed JC-1 staining to assess the changes in the MMP in HK-2 cells. The presence of green fluorescence indicates a decrease in MMP. As shown in Fig. 4A, LPS stimulation significantly reduced the MMP, while supplemented miR-338-3p alleviated the decrease in the MMP caused by LPS, indicating that LPS caused a decrease in the MMP by inhibiting miR-338-3p expression. Furthermore, an apoptosis assay showed that miR-338-3p's overexpression reduced LPS-induced apoptosis (Fig. 4B). LPS promoted the phosphorylation of p65 and p38, while overexpression of miR-338-3p inhibited the LPS-promoted phosphorylation of p65 and p38 (Fig. 5A). LPS reduced Bcl-2's mRNA and protein levels and increased Bax's mRNA and protein levels, and enhanced cleaved caspase-9 and caspase-3 levels (Fig. 5B, C). In contrast to these results, the supplementary miR-338-3p eliminated these effects (Fig. 5B, C). These data suggest that LPS led to apoptosis by reducing miR-338-3p levels.

Discussion
Sepsis is a systemic inflammatory response syndrome caused by infection that can lead to a variety of tissue and organ lesions, such as kidney damage and brain damage [23,24]. Many miRNAs are associated with inflammation [14,25]. In this study, to create an inflammatory injury model, we employed LPS to activate HK-2 cells. We observed that LPS caused HK-2 cells to become inflamed and die. We found that LPS damage in cell proliferation was reduced and the release of inflammatory cytokines was inhibited in HK-2 cells stimulated by LPS when miR-338-3p was overexpressed. Consistently, overexpression of MiR-338-3p has been shown to alleviate LPS-induced WI-38 cell damage [26]. In addition, LPSinduced apoptosis and reduced the MMP was alleviated when miR-338-3p level was increased. These data suggest that miR-338-3p has an anti-inflammatory effect in HK-2 cells.
MiR-338-3p in the serum of patients with pancreatic cancer is correlated with the neutrophil count [44].
Here, we demonstrated that miR-338-3p has a role in the inflammatory response in HK-2 cells, and that LPS increases IL-1β, IL-6, IL-8, and TNF-α expression, and impairs cell proliferation by reducing miR-338-3p levels in cells.

Conclusion
In summary, miR-338-3p alleviates inflammatory damage caused by LPS by regulating MMP, Bcl-2, Bax, P-p65, P-p38, and cleaved caspase-9 and caspase-3 levels. Thus, these results provide a new understanding of the pathological mechanism of inflammatory kidney injury and a theoretical basis for the treatment of this disease.